Approximately 55,000 solid organ transplants are performed worldwide every year. This comprises approximately 10,000 hearts, 35,000 kidneys, 16,000 livers, and 2,000 lungs. Rejection remains the most common complication following transplantation and is the major source of morbidity and mortality. There are generally recognised to be three types of organ rejection; hyperacute, acute and chronic. Hyperacute rejection occurs within 24 hours of the transplant and is readily apparent. Acute rejection is generally regarded as rejection that occurs within the first six months of transplantation, is mediated by mononuclear cells infiltrating the graft causing acute damage to graft parenchymal cells. It is usually reversed by anti-T cell cytolytic therapy. Chronic rejection, generally regarded as that occurring at least six months after transplantation, is very difficult to diagnose clinically and usually presents as a gradual vasculopathy (i.e. occlusion) of grafted vessels.
Constant vigilance is required to monitor the immune response to the grafted organ in the first 3 months, when acute rejection is most likely to occur. After kidney transplantation, raised levels of serum creatinine and urea are an indication of failing graft function, but do not specifically denote immunological damage to the graft. Nevertheless these are commonly used to detect kidney rejection, with renal biopsies used only occasionally. In contrast, monitoring the function of transplanted hearts and lungs relies entirely on histological or clinical parameters. There are no existing methods of non-invasively detecting heart or lung transplant rejection.
Thus, for patients undergoing cardiac transplantation, surveillance endomyocardial biopsies are taken at weekly intervals to 6 weeks, then at 2 weekly intervals until 3 months. In addition, any positive biopsy is followed-up by a repeat biopsy one week later to ensure that anti-rejection therapy has been successful. Patients also undergo further biopsies when clinically indicated. For example, every heart transplant patient has a minimum of 9 biopsy procedures in the first year. Lung function is routinely measured by the patients themselves using a spirometer on a daily basis. Any unexplained persistent fall in lung function will be followed up by transbronchial biopsy to confirm the diagnosis by histology. It is especially important to obtain a differential diagnosis between rejection and infection after lung transplantation. For this reason the transbronchial biopsy procedure is usually accompanied by bronchiolar lavage, which is sent for culture and bacteriological analysis.
Routine histological analysis of cardiac biopsies remain the cornerstone of management after heart transplantation and any new methods of detecting rejection are compared to the histological grading of biopsies, which are still regarded as the gold standard. A standardised nomenclature and grading system of both hearts and lungs was suggested in 1990 (1,2) and is now used by the majority of centres. However, the endomyocardial biopsy procedure is unpleasant for the patient, is associated with a small chance of complications, and is highly labour intensive and expensive. It would be of huge benefit to the patient and the hospital to have a non-invasive method to replace the endomyocardial biopsy. In theory, there are many possibilities of non-invasively detecting rejection including non-invasive monitoring of heart function such as magnetic resonance imaging (3), signal averaged electrocardiogram (4), specialised echocardiographic indices (5) and looking for markers in peripheral blood. There are two major approaches in employing blood markers; one is to exploit what is known about activation of the recipient's immune system and the second is to look for markers of graft damage.
Over the last 10 years, there has been an explosion of knowledge regarding the effect of the allograft on the immune system. Rejection is initiated by CD4+ recipient T lymphocytes recognising foreign MHC Class II molecules on antigen presenting cells in the donor graft. This initiates a cascade of cytokines that may be acting directly to damage graft parenchymal cells or maybe acting to recruit and amplify further effector mechanisms such as CD8+ T cells, macrophages and B cells. In heart transplantation, where there can be dissociation between the size of the infiltrate and the extent of cardiac haemodynamic compromise, it is thought TNF-α and nitric oxide may have negative inotropic effects on beating myocytes
Over the years there have been numerous attempts to find signs of immune activation in peripheral blood. These have included examining peripheral blood for levels of IL-2, soluble IL-2R, IL-6, IL-7, IL-8, TNF-α, IFN-γ, soluble ICAM-1, soluble MHC antigens, activated T cells and T cell populations and cytoimmunological monitoring (6). Often these are cross sectional studies and when results are pooled (i.e. comparison made between rejection and non-rejection) significant differences can be obtained. However, when one performs longitudinal studies of individual patients, the values for a particular maker vary so widely on a daily or weekly basis that sensitivities and specificities so derived are inadequate for practical use. It is clear that the immune system is highly labile for the first 3 months, when most rejection episodes occur. It will certainly be modified by augmented immunosuppression both directly (e.g. anti-thymocyte globulin binds to soluble HLA and adhesion molecules) and indirectly, by altering the balance of cell sub-populations as they recover from depletion. Taken as whole, these immune activation markers are always elevated compared to non-transplant patients, but are not reliable indicators of rejection within the individual transplant patient.
One possible reasons for the low specificity and sensitivity of the prior art markers is that they do not distinguish between rejection and infection. In order to circumvent this problem, investigations have been made to try and distinguish between donor specific and third party T cell responses as a way of assessing the state of the patient's immune system (7, 8).
We have also addressed the issue of where one would expect to find donor-specific lymphocytes, in the peripheral circulation or in the graft. To this end, we cultured lymphocytes from patients' endomyocardial biopsies and performed a limiting dilution analysis to quantify numbers of cytotoxic precursor cells with donor specific or third party specificity. At the same time, lymphocytes were cultured from patients' blood and we made a comparison of the precursor frequencies of donor specific cells found in blood and the graft. The results showed the presence of donor-reactive CD8+ T cells during rejection, but they were found almost exclusively in the graft, not in the blood. This diminishes the chances of finding specific reactivity in the peripheral circulation unless a particular sensitive assay is used. The same argument can be used for detection of circulating cytokines. It has been shown that high levels of IL-6 and soluble TNF-R1 (TNF receptor) in coronary sinus, but not aortic blood, correlated with poor coronary vasomotor tone during rejection episodes (9).
Interestingly donor specific alloantibody is produced during cell mediated acute rejection episodes in some patients (10) but this is unlikely to be a rapid enough response with which to monitor patients. An association between blood eosinophil counts and acute cardiac and pulmonary allograft rejection has been recently reported (11) whether this is specific and sensitive enough to be of practical use remains to be seen.
In the early days of heart transplantation (circa 1970's), before advent of cyclosporine, conventional serological markers of cardiac damage (lactate dehydrogenase, creatine kinase) were used as markers of graft failure. However, they lacked sensitivity and were often found to be elevated too late to reverse rejection of cardiac allografts. Troponin is a contractile regulatory complex found in striated and cardiac muscle. It consists of 3 distinct polypeptide components; troponin C, (the calcium binding element), troponin I (the actinomyosin ATPase inhibitory element) and troponin T (the tropomyosin binding element). The complex serves to regulate the calcium dependent interaction of myosin and actin and thus plays an integral role in muscle contraction.
In the 1990s, specific enzyme immunoassays have been developed against cardiac specific isoforms of troponin T and troponin I, which show little cross reactivity with the isoforms from skeletal muscle (12). With the currently commercially available kits, circulating troponin T or troponin I is only detectable in the circulation of patients with severe cardiac muscle damage such as myocardial infarction (13) or after cardiac surgery (14). Katus first reported that use of troponin T to monitor heart rejection was limited by the observation that high levels were found in the first few days after transplantation, and levels remained well above normal for 2–3 months (15). This was not related to ischaemic time and the reasons for these elevated levels are still unclear. They probably reflect low-level immunological damage caused by humoral factors (antibodies or cytokines). For this reason, the assay cannot be used to monitor rejection in the first three months, when rejection is most likely to occur. After this period, the assay does detect grade 3 or 4 rejection with a high sensitivity of 80.4% and a strong negative predictive value of 96.2% (16). It has also been used in patients six months after transplantation where rising levels are said to predict chronic rejection (17). An interesting adaptation of this assay to transplantation has been to measure levels of serum troponin T in donors; high levels correlated with occurrence of rejection in the recipients of such hearts (18), presumably reflecting damage and release of graft antigens to be recognised by the immune system.
From the above discussion, it will be apparent that it remains a continuing problem in the art to find markers which can provide an accurate and early diagnosis of acute rejection.